Device for measuring the electrical activity of biological elements and its applications

ABSTRACT

A device for measuring electrical activity of biological elements, including a substrate that has lower and upper faces and at least one through opening, the opening being delimited by a set of walls. Two plates are placed on either side of the lower and upper faces of the substrate and delimit, with the set of walls, a chamber. Each of the plates is provided, on its face lying opposite the substrate, with at least one electrode facing the opening in the substrate. Each of the plates further has at least one channel that starts inside the chamber and connects the chamber to the outside of the device, and the chamber communicates with the outside of the device only through the channels.

FIELD OF THE INVENTION

The present invention relates to a device for measuring the electricalactivity of one or more biological elements, and more especially to adevice that makes it possible to measure the electrical activity of aplurality of biological elements in parallel.

It also relates to the applications of this device.

In the above and in what follows, the term “biological element” isunderstood to mean any natural or artificial element at least part ofwhich is formed from a biological membrane or reproduces the structuraland/or functional characteristics of a biological membrane.

Thus, it may be a whole biological cell or a biological cell organelleof the type comprising a vacuole, Golgi apparatus, mitochondria,endoplasmic reticulum, lysosome, etc., a biological membrane fragment,possibly furnished with cytosolic components, an artificial lipidbilayer, such as a phosphatidyl choline or phosphatidyl glycerol film,said bilayer being provided with one or more protein pores, or else abiomimetic membrane.

The genesis and transmission of the electrical signals present inbiological cells depend on transmembrane proteins that form pores in thethickness of the plasma membranes and ensure that ions (Na⁺, Ca⁺⁺, K⁺and Cl⁻) pass through these membranes, hence their name “ion channels”.Most of these ion channels open in response to a specific disruption ofthe membrane in which they are located, which disruption may beassociated either with a change in the electrical potential of thismembrane (they are then called voltage-sensitive or voltage-dependentchannels) or with the binding of a ligand to a membrane receptor (theyare then called receptor channels).

At the present time it is known that the dysfunctioning of certain ionchannels is involved in major pathologies, among which mention may bemade of epilepsy, myotonias, cerebrospinal ataxia, high blood pressure,cardiac insufficiency, arrythmias such as ventricular quadriarrythmiasyndrome and Jervell and Lange-Nielsen syndrome, cystic fibrosis,diabetes and certain kidney disorders such as Bartter's and Gritelman'ssyndromes and pseudohypoaldosteronism type 1 (PHA-1). Likewise, certainion channels appear to be involved in the genesis and regulation ofnociceptive messages that are the cause of acute pain and chronic pain.

The device according to the invention may therefore constitute a tool ofchoice for the pharmaceutical research and especially for carrying outstudies with a view to better understanding the mechanisms responsibleat the cell level for disorders associated with an ion channeldysfunction, to identifying the sites and modes of action of drugsalready recognized as being effective in the treatment of thesedisorders and, from this, to developing new drugs that are more activeand more specific than those already available.

In particular, the device according to the invention may be applicablein the field of pharmacology, for studying antidotes and drugs againstcertain poisons or venoms (for example, scorpion venom), for the medium-or high-throughput screening of molecules that have ion channels astargets and may consequently be of therapeutic value, or of candidatedrugs so as to evaluate their effects and/or their toxicity, and also inthe field of pharmacovigilance.

It may also be used for the diagnosis of pathologies associated with anion channel dysfunction.

Moreover, ion channels of the receptor-channel type may be used as“channel sensors” in so far as any modification of their electricalactivity reflects the presence of a molecule that they are capable ofdetecting. Consequently, the device according to the invention may alsobe used in the environmental field, for example for detectingpollutants, in industries that carry out quality control checks on theirmanufacturing lines and on the products that are output thereby, such asthe agri-foodstuffs, pharmaceutical and cosmetic industries, and also inall other sectors where it is standard practice to carry outtoxicological analysis.

The device according to the invention may still be used for many otherpurposes such as, for example, for detecting living cells or cellshaving their membrane integrity preserved or, on the contrary, fordetecting dead cells or cells having lost their membrane integrity, fordetecting the release of substances from cells by exocytosis, formeasuring a variation of membrane capacitance resulting from the fusionof a cell with another cell or with a vesicle, for stimulating cells,for studying the intracellular activity of a cellular network, tissue orco-culture, for studying the response of cells to an electricalstimulation applied to another cells, or even for studyingmechanical-sensitive ion channels with the view to providing“mechanical” sensors.

PRIOR ART

It was in 1981 that Neher and Sakmann developed the technique forstudying ion channels that is still the most effective today, namely the“patch-clamp” technique (Hamill et al., Pflügers Arch., 1981,391:85-100) [1]. This technique makes it possible to check (“clamp”) thetransmembrane electrical potential differences within a plasma membranefragment (“patch”) or within a whole cell and thus obtain, directly, theion fluxes through the ion channels of this membrane patch or of thiscell.

In practice, the technique consists in applying a glass micropipette tothe plasma membrane of a cell and in creating, by suction in themicropipette, a seal having a high resistance, of the order of 1 gigaohmusually named “gigaseal”, between the end of this micropipette and themembrane patch to which the micropipette is attached (“cell-attached”configuration), it being possible to continue applying suction until theopening of this membrane patch is obtained (“whole-cell” configuration).The membrane patch can then be isolated from the rest of the cell bymechanical excision (“inside-out” or “outside-out” configurations).

It is then possible, by applying a constant electrical voltage to themembrane patch or to the cell and by recording the variations in thisvoltage, to measure the electrical activity resulting from a change ofstate (opening or closing) of the ion channels on the single isolatedmembrane patch in cell-attached or excised configuration, or on theentire cell membrane in whole-cell configuration.

Although there is no question that the patch-clamp technique, because ofits sensitivity, has allowed research to make considerable progress inthe understanding of ion channels and has given the pharmaceuticalindustry a tool for screening molecules that can act on ion channels, itturns out that this technique is not completely satisfactory.

This is because, in the first place, it has the drawback of being verysensitive to the electrical noise and vibrations coming from thesurrounding medium and, consequently, of requiring a relativelysubstantial infrastructure (anti-vibration tables, Faraday cages, etc.)designed to neutralize the interfering effects of such noise andvibration.

Moreover, it is an entirely manual method, which is quite cumbersome toimplement, especially because the glass micropipettes that it uses mustbe prepared, before each measurement, by the drawing and machining ofcapillaries, and which requires the presence of a highly trainedexperimenter, in particular for suitably positioning these micropipettesand for obtaining the gigaseal, since the suction that allows thisgigaseal to be formed is applied by mouth.

In addition, the number of specimens that can be examined per time unitis quite small and the percentage of failures high. As an indication, anexperimenter experienced in the patch-clamp technique can at best maketwenty measurements per day.

It will consequently be understood that the patch-clamp technique cannotbe used either for medium- or high-throughput pharmacological screening,as desired by pharmaceutical research, or for routine tests as isrequired for the use of ion channels as sensors.

The limitations of the patch-clamp technique as proposed by Neher andSakmann, coupled with the fact that the advances in genome research andin information technologies have demonstrated both the great diversityand great complexity of ion channels, have led to the emergence, inrecent years, of many attempts to improve this technique.

Specifically, two approaches have been explored: firstly, that aiming toautomate the measurements, while still using the same electrodes as inthe original technique (glass micropipettes), and, secondly, that aimingto carry out the measurements on electronic chips.

Thus, for example

-   -   NEUROSEARCH has developed an automatic cell recognition and        measurement apparatus for measuring eight individual cells in        separate chambers. This apparatus, which is desclosed in        WO-A-96/13721 [2], makes it possible to carry out electrical        activity measurements with high efficiency. However, since the        cells are cultured and “patched” on solid surfaces, it requires        very precise selection of the cells and the use of a system for        positioning the pipette used to establish the high-resistance        seal that is able to prevent the glass recording electrodes from        breaking.    -   SOPHION BIOSCIENCES has developed a standard substrate allowing        electrical events within biological membranes to be recorded.        This substrate, which is disclosed in WO-A-01/25769 [3], has a        plurality of sites, of square cross section, into which        electrodes are integrated. However, it turns out that in use,        these sites are ill-suited to obtaining a high-resistance seal        with the cells.    -   CENES has introduced a novel patch-clamp system for measuring        the electrical activity of whole cells. This system, which is        disclosed in WO-A-01/71349 [4], involves suspending the cells in        a liquid medium and in presenting them to an electrode, at an        air/liquid interface in a glass capillary. This system has the        advantage of eliminating the problems caused by the vibrations        emitted by the surrounding medium and of not requiring very        precise positioning of the electrode. However, it does not allow        measurements to be conducted in parallel.    -   This last company has also proposed, in WO-A-00/66329 [5], a        porous or perforated substrate, arranged in wells and on either        side of which there are electrodes. The cells are positioned on        this substrate in the form of a cell layer, so that it does not        allow measurements on individual cells to be carried out.    -   CYTION has disclosed in WO-A-99/31503 [6] a device for        positioning cells, which has the feature of including only a        single integrated measurement site per chip and of having no        structure allowing confinement of the cells. As a result, there        is a not insignificant risk of the cells spreading and of        several cells being present on the same site.    -   AXON is currently working on a draft version of a device that        uses plane silicone elastomer electrodes proposed by Yale        University in its International Application WO-A-01/59447 [7].        The first tests have shown that this device still requires        substantial improvements to be made.    -   Lastly, devices that are intended to convert ion movements        produced by ion channels into alternating electrical currents        able to be detected and measured are described in WO-A-00/25121        [8]. These devices include a substrate provided with a hole in        which an ion channel or a membrane fragment comprising one or        more ion channels is incorporated before using, and are fully        unsuitable for measuring the electrical activity of whole        biological elements such as cells or cell organelles.

The inventors were therefore given the objective of providing a devicethat can measure the electrical activity of biological elements, andespecially of cells, using the patch-clamp technique, so as to benefitfrom the advantages of this technique, in particular in terms ofsensitivity, but without, however, having the drawbacks thereof, in sucha way that it is possible with this device to make simultaneousmeasurements of cell electrical activity at a rate and with areliability that will especially permit it to be used for the medium-and high-throughput screening of potentially therapeutically usefulsubstances or for carrying out routine tests, in particular diagnosticor toxicity tests.

The inventors were also set the objective of providing a device which,while still having these advantages, is satisfactory from themanufacturing cost and operating cost standpoints.

SUMMARY OF THE INVENTION

These objectives are achieved by the present invention, which proposes adevice for measuring the electrical activity of at least one biologicalelement.

This device comprises a substantially plane substrate, which has a lowerface and an upper face and which has at least one through opening forhousing the biological element, said opening being delimited by a set ofwalls, and is characterized in that:

-   -   it comprises two substantially plane plates that are placed on        either side of the lower and upper faces of the substrate and        that delimit with said set of walls a chamber which is filled,        when the device is being used, with a liquid medium;    -   each of the plates is provided, on its face lying opposite the        substrate, with at least an electrode facing the opening in the        substrate;    -   each of the plates further has at least one channel that starts        inside the chamber and connects the latter to the outside of the        device; and    -   the chamber communicates with the outside of the device only        through said channels.

Thus, the device according to the invention comprises, for eachbiological element whose electrical activity it is desired to measure:

-   -   a chamber that is designed to be filled with a liquid medium        within which the biological element is intended to bathe in a        housing provided for this purpose, said liquid medium serving to        ensure conduction of an electric current and the survival of the        biological element, if the latter is a living element;    -   a pair of electrodes that are distributed over two plates, one        of which forms the top of the chamber while the other forms its        base, and that face the housing for the biological element,        these electrodes having the function of applying an electrical        voltage to said biological element and of recording the        variations in this voltage resulting from a change of state        (opening or closing) of its ion channels; and    -   at least two channels that connect the inside of the chamber        with the outside of the device, one of which starts at the base        of the chamber and is intended to produce a high-resistance seal        between the biological element and its housing, by creating a        vacuum in this chamber, whereas the other starts at the top of        the chamber and is intended for introducing substances into this        chamber and/or removing these substances out of said chamber.

According to a first advantageous arrangement of the invention, theopening in the substrate comprises three coaxial parts, namely an upperpart, a central part and a lower part, the upper and central partsforming a cup for housing the biological element, whereas the lower partforms a reservoir for housing a volume of liquid medium sufficient tocreate therein, by suction, a vacuum suitable for forming ahigh-resistance seal between said cup and the biological element.

As used herein, the term “high-resistance seal” refers to a seal havinga resistance of at least one hundred megaohms and advantageously of onegigaohm or above.

Preferably, the cup for housing the biological element (which forconvenience will be denoted, hereafter, only by the term “cup”) has theshape of a funnel, the frustoconical part of which corresponds to theupper part of the opening in the substrate and serves as a receptaclefor the biological element and the cylindrical part of which correspondsto the central part of this opening and serves for providing thehigh-resistance seal between this element and said cup.

This shape of cup, in the form of a funnel, has in fact proved to beparticularly suitable for confining the biological element, forrespecting its form and its integrity, especially when it is abiological cell, for ensuring its deformation or the deformation of itsmembrane during the creation of a vacuum in the subjacent reservoir and,consequently, for allowing the high-resistance seal to be obtained.

According to the invention, the dimensions of the frustoconical part ofthe cup serving as receptacle for the biological element are,preferably, matched to the dimensions of this element, thereby making itpossible to further improve its confinement and to obtain thehigh-resistance seal.

Thus, for example, when the biological element is a cell of conventionalsize, that is to say from around 10 to 30 microns in diameter(lymphocyte, CHO cell, Hela cell, HEK cell, . . . ), the frustoconicalpart of the cup—which corresponds to the upper part of the opening inthe substrate—preferably has its largest diameter between 20 and 100microns and its smallest diameter between 10 and 30 microns and has aheight of between 10 and 50 microns, whereas the cylindrical part ofthis cup—which corresponds to the central part of the opening in thesubstrate—preferably has a diameter of between 0.1 and 3 microns and aheight of 100 microns or less.

As a variant, when the biological element is a large cell, that is tosay one measuring around 0.7 to 1 mm in diameter, as is the case with axenopus ovocyte, the frustoconical part of the cup preferably has itslargest diameter between 500 microns and 1.5 mm and its smallestdiameter between 200 and 600 microns and has a height of between 300microns and 10 mm, whereas the cylindrical part of this cup preferablyhas a diameter of between 0.1 and 3 microns and a height of 100 micronsor less.

In all cases, the lower part of the opening in the substrate ispreferably cylindrical and advantageously measures between 10 and 100microns in diameter for a height of 300 to 700 microns.

According to another advantageous arrangement of the device according tothe invention, the substrate is made of one or more micromachinablematerials, micromachining being a technique that is particularly wellsuited to producing openings with dimensions ranging from one tenth of amicron to a few hundred microns.

Preferably, the substrate is based on silicon and is formed from twosilicon wafers that are placed on either side of an intermediatemembrane which they are fastened to, which intermediate membrane may bemade of an insulating material, compatible with microtechnologytechniques, or of a silicon wafer coated on its various faces by aninsulating material when it is desired to strengthen said intermediatemembrane. The insulating material may be a mineral material, such as anoxide (for example, SiO₂) or a nitride (for example, Si₃N₄), or anorganic material (for example, a polyimide).

In this situation, the upper part of the opening in the substrate (i.e.the frustoconical part of the cup) is delimited by the wall of a throughrecess made in one of the two silicon wafers that are placed on eitherside of the intermediate membrane, whereas the lower part of the openingin the substrate (i.e. the reservoir) is delimited by the wall of athrough recess made in the other of said silicon wafers.

Between the upper and lower parts of the opening in the substrate, thecentral part of this opening (i.e. the cylindrical part of the cup) is,in a simplest embodiment of the device, delimited by the wall of athrough recess made in the intermediate membrane.

However, it is also possible that the central part of the opening in thesubstrate forms a kind of micropipette that extends into the upper partof the opening in the substrate, and is delimited at the same time by afirst cylindrical wall corresponding to the wall of a through recessmade in the intermediate membrane and by a second cylindrical wallprotruding from the first cylindrical wall towards the upper face of thesubstrate. Such a configuration, the dimensions of which may beperfectly controlled, may easily be achieved by the Silicon-on-Substrate(SOI) technology, the second cylindrical wall having preferably a heightof between 1 and 30 microns for a diameter of between a few tenth of amicron and a few microns.

Quite obviously, micromachinable materials other than those previouslycited may be perfectly usable for producing the substrate, such as glassor plastics.

According to yet another advantageous arrangement of the deviceaccording to the invention, the upper face of the substrate is coveredwith a film that is made of a biocompatible and optionally flexiblematerial, and is provided with at least one through opening, thisopening being coaxial with the upper part of the subjacent opening inthe substrate, with the same geometry as it but with a larger crosssection.

This arrangement, which is particularly useful when it is desired tomeasure the electrical activity of large cells, makes it possible notonly to optimize the confinement of this type of cell but also to reducethe thickness of that part of the substrate made of the micromachinablematerial(s) and thus lower the manufacturing cost of this substratecompared with what it would be it if were entirely made ofmicromachinable material(s) such as silicon.

Preferably, the opening in the film covering the upper face of thesubstrate is frustoconical and has its largest diameter between 500microns and 1.5 mm and its smallest diameter between 200 and 600 micronsand has a height of between 300 microns and 1 mm.

Advantageously, this film is made by moulding, so that its manufacturingcost is low enough for it to be replaced with a fresh film each time thedevice is used, and is therefore made of a material capable of beingmoulded, such as a thermoplastic of the polytetrafluoroethylene (PTFE)type, or a silicone elastomer, such as a polydimethylsiloxane (PDMS).

Depending on the nature of the materials of which the film and thesubstrate are made, the film may be held in place on the substrateeither by bonding or by an adhesion effect under the effect of pressure.

According to the invention, the plates lying on either side of the lowerand upper faces of the substrate, which may be identical or different,are preferably made of an insulating material, for example aglass-epoxy, whereas the electrodes carried by these plates arepreferably plane electrodes.

The latter may especially be Ag/AgCl⁻ contacts, but contacts consistingof other oxidation-reduction pairs may also be used, such as Pt/PtCl⁻pairs.

In all cases, the electrodes are designed to be connected, after thevarious elements of the device have been assembled, to a circuit forsupplying electrical power and for measuring an electrical quantity.

According to yet another advantageous arrangement of the invention, theplate lying opposite the upper face of the substrate has two channels,namely a channel for introducing substances into the chamber and achannel for removing these substances from this chamber, whereas theplate lying opposite the lower face of the substrate has only onechannel, said channel being intended to produce the high-resistanceseal.

The channels for introducing and removing substances are designed to berespectively connected, momentarily or permanently, to capillaries thatallow them to be respectively connected to an automatic or manual liquiddelivery system and to an automatic or manual liquid suction system,these two systems being able to be connected to each other so as torecirculate said substances into the chamber.

The channel for producing the high-resistance seal is designed to beconnected, momentarily or permanently, to a capillary that allows it tobe connected to a liquid suction system.

According to the invention, these various channels may connect thechamber to the outside of the device by passing through the thickness ofthe plate in which they are located.

As a variant, they may also connect the chamber to the outside of thedevice by circulating within the plate in which they are located,substantially parallel to the faces of this plate, until reaching one ofthe edges of said plate.

According to a preferred arrangement of the device according to theinvention, the opening in the substrate, the electrodes and the channelof the plate lying opposite the lower face of the substrate are coaxial,this arrangement making it possible in particular to optimize thecreation of the high-resistance seal between the cup and the biologicalelement, the application to the latter of the electrical voltage neededto measure its electrical activity, the recording of the variations ofthis voltage and, consequently, the quality of the measurements thusmade.

According to another preferred arrangement of the device according tothe invention, the latter comprises means for sealing the chamber, whichmeans also serve for damping the electrical noise and the vibrationsemanating from the surrounding medium, and which will for convenience bedenoted hereafter only by the term “sealing means”.

These sealing means preferably consist of a first gasket, that isinserted between the substrate and the plate lying opposite the lowerface of this substrate, and of a second gasket, which may or may not beidentical to the previous one, that is inserted between the substrateand the plate lying opposite the upper face of this substrate, each ofthese gaskets being provided with at least one perforation which isarranged and dimensioned so as to circumscribe the electrode on theplate with which it is in contact.

Advantageously, these gaskets are made of a material which, apart frombeing impermeable to liquids, is flexible, slightly compressible andcapable of being moulded so that it can be produced by moulding, therebymaking it possible, on the one hand, to adapt them perfectly to theconfiguration of the substrate and, on the other hand, to manufacturethem at a low enough cost for it to be possible to replace them withfresh gaskets each time the device is used. Such a material is, forexample, an elastomer like a silicone elastomer, such as apolydimethylsiloxane.

According to yet another preferred arrangement, the substrate, theplates lying on either side of the lower and upper faces of thissubstrate and the sealing means are modules that are assembled in aremovable manner.

In this case, the device according to the invention advantageouslyincludes means for holding these modules in place in the assembledcondition.

These holding means may be means fastened to the plates lying on eitherside of the lower and upper faces of the substrate and cooperate inorder to removably fix these plates to each other, such as hinges,slideways or screwing means. They may also be means that are independentof these plates and that fit onto the edges of the stack formed by thevarious modules.

Assembly of the modules, and more particularly alignment of the axes ofthe openings in the substrate, of the electrodes carried by the plateslying on either side of the lower and upper faces of this substrate andof the perforations in the sealing means, may be facilitated by thepresence, especially on said plates, of mechanical guiding means(alignment studs, corner grooves, etc.), optical guiding means (opticalfibers passing through preformed holes), or other guiding means.

According to the invention, the device preferably allows the electricalactivity of several biological elements to be measured in parallel, inwhich case:

-   -   the substrate has a number of identical through openings        uniformly spaced apart;    -   the plates lying on either side of the lower and upper faces of        this substrate are printed circuits that are each provided with        as many electrodes as the substrate has through openings;    -   the plate lying opposite the lower face of the substrate has at        least as many channels as the substrate has through openings;        and    -   the plate lying opposite the upper face of the substrate has at        least as many channels for introducing substances and at least        as many channels for removing substances as the substrate has        through openings;        said substrate and its through openings, said plates, said        electrodes and said channels being as defined above.

According to a preferred embodiment of this device, the latter alsoincludes two identical gaskets, each provided with as many perforationsas the substrate has through openings, these gaskets and theseperforations being as defined above.

Moreover, according to this preferred embodiment, the device alsoincludes two identical clamps that fit over the edges of the stackformed by the substrate, the plates lying on either side of the lowerand upper faces of this substrate and the gaskets.

This being the case, the device according to the invention, althoughcomprising seven elements, is composed of five different modules thatcan be assembled and disassembled at will, the substrate correspondingto a first module, the plates lying on either side of the lower andupper faces of the substrate corresponding to a second and to a thirdmodule respectively, the gaskets corresponding to two examples of afourth module, while the clamps correspond to two examples of a fifthmodule.

The device according to the invention has many advantages.

This is because, while still allowing cell electrical activitymeasurements to be made according to the principles of the patch-clamptechnique, it considerably simplifies the implementation of thistechnique:

-   -   firstly, by eliminating the operations of preparing glass        micropipettes and of handling these micropipettes, the mouth        suction operation needed to obtain the gigaseal, the need to use        a microscope and equipment suitable for preventing, or at the        very least limiting, any interference due to electrical noise        and vibrations emitted in the surrounding medium; and    -   secondly, by offering the possibility of automating all or part        of these electrical activity measurements, and especially the        formation of the high-resistance seal, the delivery of        substances into chambers that are independent of one another,        the application of the electrical voltage and the recording of        the variations in this voltage, by placing the suction and        substance delivery systems and the circuit for supplying        electrical power and for measuring an electrical quantity under        the control of a computer system.

It follows that the device according to the invention makes thepatch-clamp technique available to laboratories and users that have noexperience in this field. It also follows that the device allows cellelectrical activity measurements to be made with a very satisfactorylevel of efficiency, while guaranteeing reliability, and especiallyreproducibility, of these measurements.

Moreover, it offers great flexibility of use in so far as it allowselectrical activity measurements to be made both on a plurality ofsimultaneously treated cells and on a single cell, in cell-attachedconfiguration just as well as in whole-cell configuration, and, when themeasurements are conducted in parallel, on several cells, usingparameters that may be different from one cell to another. Thus, forexample, the survival medium, the substance to be tested or detected,the concentration of this substance and the electrical voltage maydiffer from one cell to another.

In addition, it may receive, apart from whole cells, either biologicalcell organelles or fragments of biological membranes possibly furnishedwith cytosolic components, or artificial lipid bilayers, or elsebiomimetic membranes.

This operating flexibility is further enhanced when the device accordingto the invention is a modular system, since, in this case, the variousmodules may be replaced, or on the contrary reused, from one measurementto another or from one series of measurements to another.

Finally, it can be manufactured very inexpensively, especially when itis in the form of a modular system since, in this case, its manufacturedoes not include an assembly operation nor an operation to check thequality of this assembly. Similarly, the operating costs of such adevice may be very advantageous in so far as it is possible to replacesome of the modules of which it is composed, either because they areintended to be used only once, or because they have become damaged,while keeping the other ones.

Taking into account the foregoing, the device according to the inventionis capable of constituting a tool of choice for:

-   -   screening molecules for therapeutic purposes;    -   diagnosing pathologies associated with an ion channel        dysfunction;    -   detecting toxic substances, whether in the environmental field        or in the agri-foodstuffs, pharmaceutical or cosmetic        industries;    -   detecting living cells or cells having their membrane integrity        preserved or, on the contrary, detecting dead cells or cells        having lost their membrane integrity;    -   detecting the release of substances from cells by exocytosis;    -   measuring a variation of membrane capacitance resulting from the        fusion of a cell with another cell or with a vesicle;    -   stimulating cells such as neurons with a view, for example, to        studying or to promoting, or even to speeding-up, the neuronal        regeneration, new growth or plasticity;    -   studying the intracellular activity of a cellular network, of a        (organotypical) tissue or of a cellular co-culture;    -   studying the response of cells A to an electrical stimulation        applied to cells B; or even    -   studying mechanical-sensitive ion channels with the view to        providing “mechanical” sensors.

Apart from the above arrangements, the invention includes still furtherarrangements that will become apparent from the rest of the descriptionthat follows, that relates to embodiments of a device according to theinvention, and that is given by way of illustration but implying nolimitation, with reference to the appended drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a device according to theinvention, seen in exploded perspective, in an embodiment designed forthe electrical activity of nine biological cells to be measured inparallel.

FIG. 2 is a schematic representation of the device in FIG. 1, seen incross section in the plane P of this FIG. 1.

FIGS. 3 to 5 are schematic representations of a portion of a deviceaccording to the invention, in a view similar to that in FIG. 2, but forthree alternative embodiments of this device.

FIG. 6 is a schematic representation of a portion, seen in crosssection, of the substrate used in the construction of a device accordingto the invention, in a first embodiment of this substrate suitable formeasuring the electrical activity of large cells.

FIGS. 7 and 8 are schematic representations of a portion, seen in crosssection, of the substrate used in the construction of a device accordingto the invention, in two other embodiments of this substrate.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Referring firstly to FIGS. 1 and 2, these show schematically a device 10according to the invention in an embodiment designed to allow theelectrical activity of nine biological cells to be measured in parallel.

FIG. 1, which is an exploded perspective view, shows the variouscomponents of the device 10 before their assembly, while FIG. 2, whichcorresponds to a section in the plane P of FIG. 1, shows these samecomponents once they have been assembled.

As may be seen in FIGS. 1 and 2, the device 10, which is of squaregeneral shape, is composed of seven components that can be assembled ina removable manner, namely:

-   -   a first printed circuit 11, which forms the base of this device;    -   an approximately plane substrate 12 that is placed on top of the        printed circuit 11 and has the function of confining the cells        18 by means of through openings 120 in the substrate;    -   a second printed circuit 13, which is itself placed on top of        the substrate 12 and forms the cover of the device 10; this        printed circuit, together with the printed circuit 11 and the        openings 120 in the substrate 12, defines chambers 19 at the        centre of which the cells 18 whose electrical activity it is        desired to measure will be placed, one cell per chamber, which        chambers are therefore intended to be filled with an        extracellular medium or a medium acting as an extracellular        medium;    -   two perforated gaskets 4 and 5 that are inserted between the        printed circuit 11 and the substrate 12 in the case of the first        one and between this substrate and the printed circuit 13 in the        case of the second, which gaskets serve to ensure that the        device is sealed, especially between the chambers 19, and to        damp the electrical noise and the vibrations emanating from the        surrounding medium; and    -   two clamps 6 and 7 whose function is to hold the above        components in place in the assembled state.

These seven components correspond to five different modules: thus, theprinted circuit 11 corresponds to a first module or module A; thesubstrate 12 corresponds to a second module or module B; the printedcircuit 13 corresponds to a third module or module C; the gaskets 4 and5 correspond to two examples of a fourth module or module D; whereas theclamps 6 and 7 correspond to two examples of a fifth module or module E.

As shown in FIGS. 1 and 2, the substrate 12 or module B, whichrepresents the centre of the device 10, has nine through openings 120that are distributed in three rows and three columns.

The openings 120 have two functions, namely, on the one hand, to formmicrocups 40 capable of confining the cells whose electrical activity itis desired to measure and, on the other hand, to allow the formation,between these cups and these cells, of a seal with a resistance of atleast one hundred megaohms and preferably of at least one gigaohm, whena vacuum has been created within the part of the chambers 19 that liebeneath these cups.

In this way, as may be seen in FIG. 2, the substrate 12 is formed fromtwo wafers, 121 and 123 respectively, that are placed on either side ofan intermediate membrane 122 which they are fastened to, these wafersand this membrane being recessed in such a way that:

-   -   on the one hand, the cups 40 for confining the cells 18 have the        shape of funnels, the frustoconical parts 35 of which are        delimited by the wall 36 of frustoconical through recesses made        in the wafer 121 and the cylindrical parts 25 of which are        delimited by the wall 26 of cylindrical through recesses made in        the intermediate membrane 122; and    -   on the other hand, the cups 40 communicate with subjacent        cylindrical reservoirs 60, having the same axes as the cups,        which reservoirs are delimited by the wall 16 of cylindrical        through recesses made in the wafer 123 and are capable of        housing a volume of liquid medium sufficient to create therein,        by suction, the necessary vacuum for obtaining the        high-resistance seal.

As may be seen in FIG. 2, which shows, in a very schematic form, a cellbefore the high-resistance seal has been obtained (the cell on the left)and a cell after the high-resistance seal has been obtained (the cell onthe right), the frustoconical part 35 of the cups 40 serve as areceptacle for the cells 18, whereas their cylindrical part 25 servesfor producing the high-resistance seal, the latter being, in fact,obtained by deformation of the plasma membrane of said cells and byadhesion of this membrane by invagination against the wall 26 of saidcylindrical part.

To give an example, cups

-   -   whose frustoconical part 35 measures 50 μm at its largest        diameter, 30 μm at its smallest diameter and 10 μm in height and    -   whose cylindrical part 25 measures 1.5 μm in diameter and at        least 1 μm in height and    -   which sit above a cylindrical reservoir 60 having a diameter of        50 μm for a height of 450 μm, prove to be most particularly        suitable for conventional sized cells.

It will consequently be understood that the wafers 121 and 123 of thesubstrate 12 are preferably made of an easily micromachinable material,particularly silicon, the intermediate membrane 122 then beingpreferably made of a material having a high dielectric constant,suitable for being micromachined collectively (microtechnology) and ofbeing joined to a silicon wafer by bonding. Such a material is, forexample, silicon dioxide (SiO₂) or silicon nitride (Si₃N₄) when thewafers 121 and 123 are made of silicon.

The printed circuit 11 or module A serves, together with the printedcircuit 13 or module C, for applying an electrical voltage to the cells18 via the liquid medium in which they bathe. The printed circuit 11also serves to record the variations in this voltage that are induced bya change of state (opening or closing) of the ion channels of saidcells.

Thus, the printed circuits 11 and 13, the insulating supports for whichmay be made in a conventional material of the glass-epoxy type, eachcarry, on their face opposite the substrate 12, nine electrode contacts110 and 130 respectively, which are placed so that the axis of each ofthem is coincident with the axis of the cylindrical part of a cup andare designed to be connected to a circuit for supplying electrical powerand for measuring an electrical quantity (which circuit is not shown inFIGS. 1 and 2).

Thus, for example, the electrode contacts 130 of the printed circuit 13may be connected to a constant potential source, for example earth,whereas the electrode contacts 110 of the printed circuit 11 may beconnected both to an electrical generator, via an amplifier, and to anaddressing circuit suitable for sequentially collecting the electricalvoltage variations recorded by these contacts. This addressing circuitmay itself be connected, also via an amplifier, to an apparatus capableof measuring the variation of an electrical quantity, such as avoltmeter or ammeter, and which is connected to the same constantpotential source as that to which the electrode contacts 130 of theprinted circuit 13 are connected.

The operation of the circuit for supplying electrical power and formeasuring an electrical quantity and the analysis of the data collectedare, preferably, controlled by a computer system similar to that alreadyused in the patch-clamp field.

Passing through the thickness of the printed circuit 11 are also ninefluid microchannels 111, a few hundred microns in diameter, the functionof which is to create, by suction, in the cylindrical reservoirs 60, thevacuum needed to obtain the high-resistance seal. Thus, these channels111 pass through the electrode contacts 110 and are designed to beconnected, at the external face of the printed circuit 11, tocapillaries (not shown in FIGS. 1 and 2), which are themselves connectedto one or more suction systems such as those conventionally used forsucking a liquid (water pumps, for example).

Also passing through the thickness of the printed circuit 13 are fluidmicrochannels, a few hundred microns in diameter, but there are eighteenof these distributed as nine channels 131 whose function is to introducesubstances into the chambers 19 and nine channels 132 whose function isto remove these substances from said chambers. The channels 131 and 132may also be used to recirculate the substances into the chambers 19.

In the embodiment of the device according to the invention shown in FIG.1, the channels 131 and 132 lie on either side of the electrode contacts130. Like the channels 111, the channels 131 and 132 are designed to beconnected to capillaries (not shown in FIGS. 1 and 2) that arethemselves connected to one or more liquid delivery systems(microsyringes, micropumps, etc.) and to one or more liquid suctionsystems or to one or more systems suitable for recirculating thesubstances into the chambers 19.

Here again, the operation of these various delivery, suction orrecirculation systems is preferably controlled by a computer system.

As mentioned above, two gaskets 4 and 5, that correspond to two examplesof module D, are inserted between the printed circuit 11 and thesubstrate 12 in the case of the first gasket, and between this substrateand the printed circuit 13 in the case of the second, for the purpose ofsealing the device, especially between the chambers 19, and of dampingthe electrical noise and the vibrations emanating from the surroundingmedium.

To do this, these gaskets are provided with circular perforations, 140and 150 respectively, that have a diameter slightly greater than boththe largest diameter of the cups 40 and the diameter of the cylindricalreservoirs 60, and the axis of which is coincident with the axis ofthese cups and of these cylindrical reservoirs.

Moreover, these gaskets, which may be only a few microns in thickness,are made of a liquid-impermeable, slightly compressible and possiblyflexible material, which is preferably an elastomer like a siliconeelastomer, such as a polydimethylsiloxane (PDMS).

Moreover, they are preferably manufactured by moulding so that, on theone hand, they are perfectly matched to the configuration of thesubstrate 12 and, on the other hand, their manufacturing cost is lowenough for them to be able to be replaced with new gaskets each time thedevice 10 is used.

The clamps 6 and 7, that correspond to two examples of module E, havethe function of holding the modules A, B, C and D in place once theyhave been assembled. Having a U-shaped cross section and a lengthsubstantially equal to the edges of the printed circuits 11 and 13, theycan be fitted onto the edges of the stack formed by said modules afterslight pressure has been-applied to this stack.

Referring now to FIGS. 3 to 5, these show a portion of a device 10, inthree embodiments that differ from one another and from the embodimentshown in FIGS. 1 and 2, by the arrangement of the fluid microchannels131 and 132.

In the embodiment shown in FIG. 3, the channels 131 and 132 both passthrough the electrode contact 130.

In the embodiment shown in FIG. 4, only one of these channels, forexample the channel 131, passes through the electrode contact 130, thechannel 132 being placed laterally with respect to this electrodecontact, whereas in the embodiment shown in FIG. 5 the two channels 131and 132 are placed laterally and on the same side as said electrodecontact.

FIG. 6 shows a portion of a substrate 12 intended to be used in theconstruction of a device 10, in an embodiment designed to measure theelectrical activity of large cells, that is to say cells measuringaround 0.7 to 1 mm in diameter.

In this embodiment, the upper face of the substrate 12 is covered with afilm 126 which is made of a biocompatible and optionally flexiblematerial, such as a PDMS, a resin or a thermoplastic, and which isprovided with through openings 127, coaxial with the openings 120 in thesubstrate. The dimensions of the openings 127 of the film are such thatthe cups comprise, in this case, two superposed frustoconical parts,namely a first part, which corresponds to the recesses 35 made in thewafer 121, and a second part, which corresponds to the openings 127 inthe film 126.

To give an example, cups:

-   -   whose first frustoconical part measures 500 μm at its largest        diameter, 300 μm at its smallest diameter and 450 μm in height,    -   whose second frustoconical part measures 1 mm at its largest        diameter, 600 μm at its smallest diameter and 1 mm in height,        and    -   whose cylindrical part measures 1.5 μm in diameter and less than        1 μm in height, have given excellent results.

Here again, it is preferable for the film 126 to be produced by mouldingso that its manufacturing cost is low enough for it to be able, like thegaskets 4 and 5, to be replaced with a fresh film each time the device10 is used.

FIGS. 7 and 8 also show a portion of a substrate 12 intended to be usedin the construction of a device 10, but in which a cylindrical wall 50protudes from the wall 26 of each of the cylindrical recesses 25provided in the intermediate membrane 122 so as to form with the lattera kind of micropipette which extends into the upper part 35 of theopenings 120 in the substrate.

In the embodiment shown in FIG. 7, the intermediate membrane 122 isconstituted by only one material, preferably a micromachinable materialwith a high dielectric constant, whereas in the embodiment shown in FIG.8, the intermediate membrane 122 is strengthened by a silicon wafer andis therefore constituted by a silicon wafer 51 coated on all its facesby a layer 52 of a material with a high dielectric constant.

In both cases, the cylindrical wall 50 is constituted by the samematerial as the one forming the wall 26 of the cylindrical recesses 25provided in the intermediate membrane 122.

As an example, when the device 10 is intended to be used for measuringthe electrical activity of cells of a conventional size, then substrate12 as it is shown in FIG. 2 may be based on silicon, for exampleproduced by a process comprising the following steps:

a) preparation of the wafer 123 by:

-   -   polishing, on both sides, a first silicon wafer until a        thickness of about 450 μm is obtained,    -   depositing, on both faces of the silicon wafer thus polished, a        layer of SiO₂,    -   producing, in the thickness of one of the two SiO₂ layer,        cylindrical openings approximately 1.5 μm in diameter and    -   producing, in the thickness of the other SiO₂ layer and of the        silicon wafer, cylindrical recesses 50 μm in diameter by deep        chemical etching, taking measures to ensure that the axis of        these recesses is coincident with that of the openings made in        the previous step;

b) fastening, by wafer bonding, the wafer 123 thus obtained to a secondsilicon wafer polished beforehand on both sides, this second wafer beingintended to form the future wafer 121;

c) polishing of this second silicon wafer until a thickness of 10 μm isobtained;

d) deposition, on this second silicon wafer, of a layer of SiO₂ orSi₃N₄; and

e) production, in the thickness of the Si₃N₄ layer and of the secondsilicon wafer, of frustoconical recesses.

When the device 10 is intended to be used for measuring the electricalactivity of large cells, then the substrate 12 as it is shown on FIGS. 2and 6 may be based on silicon, for example produced by a processcomprising the following steps:

a) preparation of the wafer 123 as described above;

b) preparation of the wafer 121 by:

-   -   polishing, on both sides, a second silicon wafer until a        thickness of approximately 450 μm is obtained,    -   depositing, on both sides of the silicon wafer thus polished, a        layer of SiO₂ or Si₃N₄,    -   producing, in the thickness of one of the two SiO₂ or Si₃N₄        layers, cylindrical openings approximately 500 μm in diameter,        and    -   producing, starting from these openings, frustoconical recesses        in the thickness of the silicon wafer by deep chemical etching;

c) fastening, by wafer bonding, the wafer 121 thus obtained to the wafer123, and then, optionally;

d) bonding or adhesion by simply pressing a film 126 onto the wafer 121.

The use of the device 10 is extremely simple.

Indeed, after having deposited a module D on a module A, and then amodule B matched to the size of the cells whose electrical activity itis desired to measure, on the module D, the cups of the module B arefilled with an extracellular medium or a medium that can be used as anextracellular medium, this medium having to be capable of ensuring bothconduction of an electrical current and the survival of the cells.

The air present in the cylindrical reservoirs 60 is sucked out by meansof the fluid microchannels 111, in order to allow the extracellularmedium to flow into these reservoirs. This operation is repeated untilthe cylindrical reservoirs 60, and then the cups 40, have beencompletely filled with said extracellular medium.

In the case of large cells measuring around 0.7 to 1 mm in diameter suchas xenopus ovocytes, they are deposited in the cups 40, for example bymeans of a pipette, with one cell per cup, and again suction is applied,via the fluid microchannels 111, in the cylindrical reservoirs 60 inorder to obtain a high-resistance seal between each of the cells withthe cup in which it is placed and then, possibly, to rupture the plasmamembrane fragment thus sealed, if it is desired to work in whole-cellconfiguration.

Once this seal has been obtained for all the cells, the module B iscovered with a module D, then the module D is covered with a module Cand, after slight pressure has been applied to the resulting stack ofmodules A, B, C and D, the modules E are then attached.

In the case of cells of conventional size such as lymphocytes or CHOcells, a suspension of cells, for example containing 105 cells/ml ofsuspension, is distributed in the whole substrate 12. A suction isapplied, via the fluid microchannels 111, in the reservoirs 60 in orderto obtain a high-resistance seal between each of the cells with the cupin which it is placed and then, possibly, to rupture the plasma membranefragment thus sealed. A washing of the substrate 12 allows to remove thecells that are not sealed. The device is closed as previously described.

Then, the device 10 is connected to the circuit for supplying electricalpower and for measuring an electrical quantity and the electricalactivity of the cells is measured exactly as in the original patch-clamptechnique, except that the substances to be tested or detected areintroduced into the chambers 19 via the fluid microchannels 131.

When the tests have been completed, the device 10 can be easilydismantled for the purpose of using it again. All that is required is,after the circuit for supplying electrical power and for measuring anelectrical quantity has been disconnected, that the modules E be removedso as to be able to separate the modules A, B, C and D from one another.The modules A, B and C may be reused after they have been suitablywashed, while the modules D are thrown away to be replaced with freshmodules for subsequent use of said device.

The invention is no way limited to the embodiments that have beendescribed. Thus, for example, the device according to the invention iscapable of being adapted to the parallel measurement of the electricalactivity of a much larger number of cells, for example 1000 cells, oreven more, in which case the substrate 12 is designed to accommodatethis number of cells.

Furthermore, the device according to the invention may be coupled to orintegrated into other analysis systems such as systems allowing thedetection of fluorescence or luminescence signals. In such a case, theprinted circuit 13 is made of a transparent material. Such a coupling orintegration allows to relate in real time an electrical activity to anionic signalisation and thus to obtain a response which is at the sametime structural and functional, that is to say a dynamic response.

BIBLIOGRAPHIC REFERENCES

-   -   [1] HAMILL et al. Pflügers Arch., 1981, 391:85-100    -   [2] WO-A-96/13721    -   [3] WO-A-01/25769    -   [4] WO-A-01/71349    -   [5] WO-A-99/66329    -   [6] WO-A-99/31503    -   [7] WO-A-01/59447    -   [8] WO-A-00/25121

1. A device for measuring electrical activity of at least one biologicalelement, comprising: a substantially plane substrate, which has a lowerface and an upper face and which has at least one through opening forhousing the biological element, said opening being delimited by a set ofwalls; two substantially plane plates placed on either side of the lowerand upper faces of the substrate and that delimit with the set of wallsa chamber that is filled, when the device is being used, with a liquidmedium; wherein each of the plates is provided, on its face lyingopposite the substrate, with at least one electrode facing the openingin the substrate; wherein each of the plates further has at least onechannel that starts inside the chamber and connects the chamber tooutside of the device; and wherein the chamber communicates with theoutside of the device only through the channels.
 2. A device accordingto claim 1, wherein the opening in the substrate comprises an upperpart, a central part, and a lower part that are coaxial, the upper andcentral parts forming a cup for housing the biological element, whereasthe lower part forms a reservoir for housing a volume of liquid mediumsufficient to create therein, by suction, a vacuum suitable for forminga high-resistance seal between the element and the cup.
 3. A deviceaccording to claim 2, wherein the upper part of the opening in thesubstrate is of frustoconical shape, whereas the central part of theopening is of cylindrical shape.
 4. A device according to claim 3,wherein the upper part of the opening in the substrate has its largestdiameter between 20 and 100 microns and its smallest diameter between 10and 30 microns and has a height of between 10 and 50 microns, whereasthe central part of the opening has a diameter of between 0.1 and 3microns and a height of 100 microns or less.
 5. A device according toclaim 3, wherein the upper part of the opening in the substrate has itslargest diameter between 500 microns and 1.5 mm and its smallestdiameter between 200 and 600 microns and has a height of between 300microns and 10 mm, whereas the central part of the opening has adiameter of between 0.1 and 3 microns and a height of 100 microns orless.
 6. A device according to claim 2, wherein the lower part of theopening in the substrate is cylindrical and measures between 10 and 100microns in diameter for a height of 300 to 700 microns.
 7. A deviceaccording to claim 1, wherein the substrate comprises one or moremicromachinable materials.
 8. A device according to claim 2, wherein thesubstrate comprises silicon.
 9. A device according to claim 1, whereinthe substrate is formed from two silicon wafers that are placed oneither side of an intermediate membrane to which the two silicon wafersare fastened.
 10. A device according to claim 9, wherein theintermediate membrane comprises an insulating material or a siliconwafer coated on its various faces by an insulating material.
 11. Adevice according to claim 10, wherein the insulating material comprisessilicon dioxide or silicon nitride.
 12. A device according to claim 9,wherein the upper part of the opening in the substrate is delimited bythe wall of a through recess made in one of the silicon wafers that areplaced on either side of the intermediate membrane, whereas the lowerpart of the opening of the substrate is delimited by the wall of athrough recess made in the other of the silicon wafers.
 13. A deviceaccording to claim 9, wherein the central part of the opening in thesubstrate is delimited by the wall of a through recess made in theintermediate membrane.
 14. A device according to claim 9, wherein thecentral part of the opening in the substrate is delimited by a firstcylindrical wall corresponding to the wall of a through recess made inthe intermediate membrane and by a second cylindrical wall protrudingfrom the first cylindrical wall towards the upper face of the substrate.15. A device according to claim 2, wherein the upper face of thesubstrate is covered with a film made of a biocompatible material and isprovided with at least one through opening, the opening being coaxialwith the upper part of the opening in the substrate, with a samegeometry but with a larger cross section.
 16. A device according toclaim 15, wherein the opening in the film is frustoconical and has itslargest diameter between 500 microns and 1.5 mm and its smallestdiameter between 200 and 600 microns and has a height of between 300microns and 1 mm.
 17. A device according to claim 1, wherein the plateslying on either side of the lower and upper faces of the substratecomprise an insulating material and the electrodes carried by the platesare plane electrodes, especially Ag/AgCl⁻ contacts.
 18. A deviceaccording to claim 1, wherein the plate lying opposite the upper face ofthe substrate has two channels.
 19. A device according to claim 1,wherein the plate lying opposite the lower face of the substrate hasonly one channel.
 20. A device according to claim 18, wherein the twochannels of the plate lying opposite the upper face of the substratepass through the thickness of the plate.
 21. A device according to claim1, wherein the one channel of the plate lying opposite the lower face ofthe substrate passes through the thickness of the plate.
 22. A deviceaccording to claim 18, wherein the two channels of the plate lyingopposite the upper face of the substrate circulate within the thicknessof the plate, substantially parallel to the faces of the plate, untilreaching one of edges of the plate.
 23. A device according to claim 19,wherein the one channel of the plate lying opposite the lower face ofthe substrate circulates within the thickness of the plate,substantially parallel to the faces of the plate, until reaching one ofedges of the plate.
 24. A device according to claim 1, wherein theopening in the substrate, the electrodes carried by the plates lying oneither side of the lower and upper faces of the substrate, and thechannel of the plate lying opposite the lower face of the substrate arecoaxial.
 25. A device according to claim 1, further comprising means forsealing the chamber and for damping electrical noise and vibrationsemanating from a surrounding medium.
 26. A device according to claim 25,wherein the means for sealing the chamber and for damping the electricalnoise and the vibrations emanating from the surrounding medium comprisesa first gasket inserted between the substrate and the plate lyingopposite the lower face of the substrate, and a second gasket insertedbetween the substrate and the plate lying opposite the upper face of thesubstrate, each of the first and second gaskets being provided with atleast one perforation arranged and dimensioned to circumscribe theelectrode on the plate with which it is in contact.
 27. A deviceaccording to claim 25, wherein the substrate, the plates lying on eitherside of the lower and upper faces of this substrate, and the means forsealing the chamber and damping the electrical noise and the vibrationsemanating from the surrounding medium are modules that are assembled ina removable manner.
 28. A device according to claim 27, furthercomprising means for holding the substrate, the plates that lie oneither side of the lower and upper faces of the substrate, and the meansfor sealing the chamber and damping electrical noise and vibrationsemanating from a surrounding medium, in place in an assembled condition.29. A device according to claim 1, configured for measuring electricalactivity of plural biological elements in parallel; wherein thesubstrate comprises a plurality of identical through openings uniformlyspaced apart; wherein the plates lying on either side of the lower andupper faces of the substrate are printed circuits that are each providedwith as many electrodes as the substrate has through openings; whereinthe plate lying opposite the lower face of the substrate has at least asmany channels as the substrate has through openings; and wherein theplate lying opposite the upper face of the substrate has at least asmany channels for introducing substances and at least as many channelsfor removing substances as the substrate has through openings.
 30. Adevice according to claim 29, comprising two identical gaskets eachprovided with as many perforations as the substrate has throughopenings.
 31. A device according to claim 30, comprising two identicalclamps that fit over edges of a stack formed by the substrate, theplates lying on either side of the lower and upper faces of thesubstrate, and the gaskets.